It is becoming increasingly valuable to be able to measure enzymatic activity in living organisms. Since enzymes mediate all major cellular functions, such as metabolism, respiration and immune system response, analysis of enzymatic activity plays an important role in understanding cellular function, and diagnosing and treating disease.
Fluorescence-based analytical methods provide a sensitive means to investigate cellular enzymatic activity. These techniques are especially amenable to recently-developed microscopic imaging technologies which allow two-dimensional images to be collected, analyzed with computer software and stored in a digital format. The research in this arena focuses on the spatial and temporal characteristics of enzymatic activity in single cells and single microorganisms. Investigations of single cells and single microorganisms allows better understanding of the bulk data collected when analyzing populations of cells or organisms. Even further, the real possibility of identifying cells or organisms possessing specific characteristics, desired or undesired, will prove to be useful in medicine, chemistry and environmental research.
In some prior work by Bronk, Powers and Gores at the Mayo Medical School and reported in the journal Analytical Biochemistry, Vol. 210, pp. 219-25 (1993), a baseline emitter was chemically attached to a first carrier particle and the active component was chemically attached to a second carrier particle. In this work the baseline emitter was rhodamine, which was attached to the first carrier particles, made of dextran. The active component was glycine-7-amino-4-methylcoumarin-3-acetic acid ("glycine-AMC-3-acetic acid"), a non-fluorescer, which was attached to the second carrier particles, also dextran, by a polyethylene glycol ("PEG") bridge. A mixture of the two types of carrier particles was microinjected into cultured rat hepatocytes to measure aminopeptidase activity. When the active component particles are exposed to the hepatocyte environment, the attack of the aminopeptidase upon the glycine attachment to the glycine-AMC-3-acetic acid-PEG-dextran liberates the fluorescent compound AMC-3-acetic acid-PEG-dextran. The emission intensity of rhodamine-dextran remained constant over time, but the emission intensity of AMC-3-acetic acid-PEG-dextran increased in a linear fashion, indicating the proteolytic cleavage of the glycine-AMC bond. The measurement of the emission intensity was achieved through the known technique of single excitation dual emission wavelength ratio technique ("SEDERT"). As it turned out, the first and second carrier particles co-localized in the cytosol, that is, the cytoplasm of the cell less the mitochondria and endoplasmic reticulum components, as indicated by the diffuse fluorescence of each across the cytosol. This allowed the fluorescent ratio of the active component to the baseline emitter to be compared, thus measuring proteolysis. However, had the first and second carrier particles not co-localized, the ratio technique would not have been possible.
In the field of fluorescence-based biological assays, several difficulties are encountered, but the major difficulty involves delivery of the fluorescent probe to the targeted location. It is well known to use acetoxymethyl esters to transport fluorescent ion and enzyme probes through the cell membrane, but these systems often require use of dimethylsulfoxide ("DMSO") as the carrier-reagent. Once through the membrane, the intercellular esterases transform the probe into an active state. However, not all probes are amenable to this type of derivatization, and long incubation times are often required to deliver a measurable amount across the membrane. Once inside the cell, unwanted localization or undesirable binding of the fluorescent probe is common. This localization may be due to a variety of factors, including solubility and electrical potential characteristics of the probe relative to membranes and cell organelles. Similarly, non-uniformities in the source intensity of the excitation source result in fluctuations in the measured fluorescence, and detract from quantitation of the cellular processes. Without a baseline against which to compare the active probe intensity, these fluctuations impose huge obstacles to an otherwise convenient test method.